Biological Functions of Nitric Oxide (NO)
Nitric oxide (NO) is a ubiquitous molecule with various biological functions, based mainly on its vasodilatory effect (Ignarro et al., 1989).
NO is a diffusible gas that can be classified as a chemical messenger, capable of:                modulating the activity of specific NO-binding enzymes: for instance guanylate cyclase, which induces the formation of the second cGMP messenger (cGMP causes vasodilation in vascular smooth muscle);        binding and modifying enzymes and ion channels (nitrosylation), which thereby change their activity.        
NO is produced by the enzyme NO synthase, which degrades arginine and citrulline, releasing an NO molecule (Rajfer et al., 1992).
There are various isoforms of NO synthase, which can be classified schematically according to their basal activity.
NO is rapidly degraded/inactivated; it is therefore, basically a “locally acting” second messenger it differs from classic second messengers in that it can perform its effects also on cells (Fulton et al., 2001; Furchgott et al., 1980).
Biological Role of NO in the Physiology of Penile Erection
Penile erection is a physiological phenomenon resulting from a haemodynamic event and a neurogenic event.
It is a haemodynamic event in that the degree of erection depends on the balance between arterial inflow (pudendal arteries) and venous outflow (superficial veins, deep dorsal vein, and the veins of the corpora cavemosa).
It is a neurogenic event in that the penis is flaccid when the arterial and venous inflow are equally balanced; it is tumescent when the arterial inflow increases and the venous outflow decreases.
In the flaccid penis the main system responsible for maintaining vasoconstriction and contraction of the smooth muscle of the corpora cavemosa is the sympathetic nervous system (low blood flow to the penis); in the erect penis the system that maintains dilatation of the arteriole and relaxation of the trabecular smooth muscle is the parasympathetic nervous system.
Biochemistry of Penile Erection
As indicated above, at the time of erection the muscles of the corpora cavemosa of the penis are relaxed and, at the same time, the arterial flow increases and the venous blood flow decreases.
This mechanism is regulated by a nerve component, which makes erection a neurovascular process involving a series of transmitters and modulators.
A central role in this process is performed by the nitric oxide (NO) molecule, produced in its capacity as a mediator in response to stimulation from non-adrenergic, non-cholinergic (NANC) nerve fibres present within the smooth muscle of the corpora cavemosa of the penis and also present in the arteries and veins (Palmer et al., 1987).
The NO mediator is able to cause the corpora cavemosa to relax, a fundamental event for the purposes of erection.
During the penile erection process the production of NO by NO synthase of endothelial origin (eNOS) increases, which has a vasodilatory effect on the vascular endothelium.
This happens in physiological conditions, in which the endothelium is not affected by any pathology.
In pathological conditions, however, the biochemical pathways for NO production are disrupted (Cayatte et al., 1994). This causes biochemical changes responsible for the altered homeostasis that causes penile erection disorders.
It is believed, moreover, that during erection the primary factor involved is production of NO by the isoform of neuronal NO synthase (nNOS) following nerve stimulation, causing a transient increase in blood flow and increased blood circulation in the penis and sinusoidal spaces.
Next, a further, substantial production of NO by eNOS occurs, making an effective erection possible.
The NO mediator, which arrives by diffusion into the smooth muscle cells of the corpora cavemosa, activates the enzyme guanylate cyclase (GC), which is in turn responsible for producing a cyclic second messenger, cGMP.
These cyclic nucleotides are subjected to a process of hydrolysis by phosphodiesterase (PDE) isoenzymes, and, particularly in the corpora cavemosa of the penis, the PDE 5 isoform (pharmacological target of Viagra-like drugs that inhibit phosphodiesterase) (Moncada et al., 1993).
Over the last few years, however, it has become increasingly clear that many of the cellular actions of NO take place also in cGMP-independent mode; in-vivo reactions have been described between NO and oxygen, iron, the haem group, proteins containing thiol groups, DNA and some amines subject to S-nitrosylation (Marietta et al., 1993).
In view of the above, a system that increases the availability of NO is highly desirable in many pathophysiological conditions, particularly where there is sexual dysfunction.
In fact, in normal conditions, the release of NO onto the endothelial walls of the blood vessels promotes the vasodilatory effect, which causes penile erection, among other things.
Therefore, to improve the process of penile erection, particularly in cases where it is impaired, it is highly desirable to provide NO, chiefly in topical form, in order to produce vasodilatory effects similar to those occurring in physiological conditions.
It is also important that, when needed, this effect of NO is immediate but also lasts for some time.
In general, the administration of NO can be useful in many pathophysiological conditions where vasodilatory and, in particular, peripheral action is required, for instance because of endothelial dysfunction.
Furthermore, the administration of NO can be beneficial also in situations where antimicrobial action is needed.
Nonetheless, to provide bioavailable NO, the molecule has to be stabilised because it is too reactive to reach the desired target within the body. Moreover, its use is made difficult by its very short half-life and its chemical nature as an unstable and reactive gas.
There are various known systems for releasing NO, in which it is produced or released at the time of use.
Known release methods typically involve polymers and small molecules such as S-nitroso-N-acetylpenicillamine and S-nitrosocysteine (CysNO), which release NO into the body.
These methods are not, however, able to provide sufficient quantities of NO for long periods of time or in a controlled manner since S-nitrosothiols, for example S-nitrosoglutathione (GSNO) and S-nitrosocysteine—natural NO donors—are particularly unstable in aqueous environment and in biological fluids (Moncada et al., 1993). In fact, formulations based on NO donors in solution typically decompose quantitatively within a few hours (Broniowska et al., 2012). Systems for the release of NO in aqueous media are disclosed in WO2010/086637, US2015/086651, WO2012/052561, U.S. Pat. No. 7,048,951.
Various systems for the topical administration of NO are also known.
For example, US2013089629 describes a kit for treating onychomycosis using NO comprising an acidifying agent to be used as pretreatment and a composition of nitrite and at least one polysaccharide with a sufficient amount of ascorbic acid to produce NO for use as a treatment.
U.S. Pat. No. 7,048,951 describes a system for the topical release of NO comprising an ointment composed of a non-aqueous medium and sodium nitrite, ascorbic acid and maleic acid as active ingredients which, when applied to the skin, release NO.
WO2008153762 describes a composition comprising an S-nitrosothiol, for example GSNO, in a pharmaceutical formulation that permits its long-term storage before administration to the patient.
WO1999044622 describes a topical formulation comprising nitric oxide, wherein the nitric oxide is generated when an acidifying agent and a nitric oxide donor come into contact at the site of action. The composition is used in the treatment of viral skin infections.
WO201102265 also describes gels for topical release of NO; such gels include functionalised polysiloxane macromolecules.
Some of these methods are also aimed at treating sexual dysfunction.
For example, US20130059017 describes a composition for topical delivery of nitric oxide to treat sexual dysfunction comprising a first phase comprising lecithin and nitric oxide and a second phase comprising an emulsifier, such that the nitric oxide is entrapped, for example through the formation of vesicles, and therefore remains intact. The second phase can also comprise polyglycols.
Various systems and solutions for releasing NO and therefore described in the prior art, intended in particular to deal with the problem of this compound's instability.
There is still a need for stable compositions, particularly for topical use, capable of releasing NO, when required, effectively and immediately, but at the same time capable of ensuring that NO is released for a sufficiently long period.
This problem is particularly heartfelt in the context of treatment for erectile dysfunction, in which immediate action that also lasts for a certain length of time is particularly desirable.